Posttranslational addition of hydrophobic moieties (fatty acid acylation and prenylation) is a functionally essential modification for many proteins involved in intracellular signalling pathways (Olsen, et al., Biochemistry, 29, 2623-2634 (1990); McIlhinney, et al., J. Trends Biochem. Sci., 15, 387-391 (1990); Gordon, et al., J. Biol. Chem., 266, 8647-8650 (1991); Glomset, et al., Trends Biochem. Sci., 15, 139-142 (1990); Maltese, et al., FASEB J., 4, 3319-3328 (1990); Rine, et al., New Biologist, 2, 219-226 (1990); Der, et al., Cancer Cells 3, 331-340, (1991); Sinensky, et al., BioEssays, 14, 25-31 (1992)).
Farnesyl:protein transferase (FPTase) catalyzes the transfer of a hydrophobic farnesyl group (C.sub.15) from farnesyl diphosphate to a specific C-terminal cysteine residue of a protein substrate, forming a thioether bond and displacing inorganic pyrophosphate in the process (Manne, et al., Proc. Natl. Acad, Sci. USA, 87, 7541-7545 (1990); Schaber, et al., J. Biol. Chem., 265, 14701-14704 (1990); Reiss, et al., Cell, 62, 81-88 (1990)). Short peptides (.gtoreq.4 residues) containing a C-terminal consensus recognition sequence can also serve as farnesylation acceptor substates (Reiss, et al., Cell, 62, 81-88 (1990); Moores, et al., J. Biol. Chem., 266, 14603-14610 (1991)). Interest in FPTase has intensified because farnesylation is required for membrane association and biological function of ras-encoded proteins (Willumsen, et al., EMBO J. 3, 2581-2585 (1984); Hancock, et al., Cell, 57, 1167-1177 (1989); Jackson, et al., Proc. Natl. Acad. Sci. USA, 87, 3042-3046 (1990)), mutant forms of which play a causitive role in over 20% of all human cancers, including greater than 50% of pancreatic and colon tumors (Bos, Molecular Genetics in Cancer Diagnosis (Cossman, J., Ed.), Elsevier Science Publishing Co., New York, pp 273-288 (1991)).
Inhibition of FPTase represents a possible method for preventing relocation of mutant Ras from the cytosol to the membrane, thereby blocking its cell transforming function. Enzymological studies and the search for potent, specific inhibitors led to consideration of alternatives to the existing stopped-point assays for FPTase activity, which are labor intensive and generate radioactive waste (Reiss, et al., Methods: A Companion to Methods in Enzymology, 1, 241-245 (1990)).
Study of the transferase enzymes that catalyze modifications for many proteins involved in intracellular signalling pathways has been hampered by troublesome stopped-point methods for assaying their activity. It is therefore an object of this invention to develop a continuous fluorescence assay for one of these enzymes, farnesyl:protein transferase. This assay utilizes a feature found in hydrophobic modification reactions which is the increase in hydrophobicity about the reaction center of the acceptor substrate that occurs following conversion of substrate to product. In comparison to the stopped point assays as found in the art, the assay of this invention is more accurate, less time-consuming, and does not produce radioactive waste.